Microfluidic system and method for arranging objects

ABSTRACT

Microfluidic systems and methods for arranging a set of objects. In an exemplary method, the set of objects may be transported in carrier fluid along a microfluidic channel structure having a reformatting zone including an object-accessible region and at least one object-excluding region. A portion of the carrier fluid may be moved from the object-accessible region to the at least one object-excluding region in an upstream section of the reformatting zone, to reduce a spacing of objects of the set. The portion of the carrier fluid may be directed into the object-accessible region from the at least one object-excluding region in a downstream section of the reformatting zone, to increase a spacing of objects of the set. The steps of moving and directing in combination may increase the spacing between objects disproportionately for a subset of the objects that are closest to one another.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application is based upon and claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 62/580,338, filedNov. 1, 2017, which is incorporated herein by reference in its entiretyfor all purposes.

INTRODUCTION

Microscopic objects, such as beads, cells, or droplets, can be arrangedin a stream by a microfluidic system to enable detection or furthermanipulation for an assay. However, the spacing of objects within thestream is usually stochastic. As a result, some of the objects may be invery close proximity to one another, which can make the assay moredifficult to perform and may introduce error into assay results.

SUMMARY

The present disclosure provides microfluidic systems and methods forarranging a set of objects. In an exemplary method, the set of objectsmay be transported in carrier fluid along a microfluidic channelstructure having a reformatting zone including an object-accessibleregion and at least one object-excluding region. A portion of thecarrier fluid may be moved from the object-accessible region to the atleast one object-excluding region in an upstream section of thereformatting zone, to reduce a spacing of objects of the set. Theportion of the carrier fluid may be directed into the object-accessibleregion from the at least one object-excluding region in a downstreamsection of the reformatting zone, to increase a spacing of objects ofthe set. The steps of moving and directing in combination may increasethe spacing between objects disproportionately for a subset of theobjects that are closest to one another. The method may produce a moreuniform spacing of objects and/or a lower incidence of objects in closeproximity to one another.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an exemplary microfluidic system forarranging objects, in accordance with aspects of the present disclosure.

FIG. 2 is a sectional view of the microfluidic system of FIG. 1, takengenerally along line II-II of FIG. 1 at a position upstream of areformatting zone of the system.

FIG. 3 is another sectional view of the microfluidic system of FIG. 1,taken generally along line III-III of FIG. 1 at a position intersectingthe reformatting zone of the system.

FIG. 4 is a fragmentary view of the system of FIG. 1 in the presence ofa pair of objects (“A” and “B”) that are in very close proximity to oneanother upstream of the reformatting zone and illustrating how thecenter-to-center distance (the spacing) between the objects may bechanged by traveling through the reformatting zone of the system(compare spacing S1, spacing S2, and spacing S3).

FIG. 5 is another fragmentary view of the system of FIG. 1, taken as inFIG. 4, except with the pair of objects farther apart than in FIG. 4upstream of the reformatting zone.

FIG. 6 is yet another fragmentary view of the system of FIG. 1, taken asin FIG. 4, except with the pair of objects farther apart than in FIG. 5upstream of the reformatting zone.

FIG. 7 is still another fragmentary view of the system of FIG. 1, takenas in FIG. 4, except with the pair of objects farther apart than in FIG.6 upstream of the reformatting zone.

FIG. 8 is a histogram showing an exemplary distribution of thecenter-to-center distances (the spacing) between objects before theobjects have been passed through the reformatting zone of themicrofluidic system of FIG. 1, in accordance with aspects of the presentdisclosure.

FIG. 9 is a histogram showing an exemplary distribution of thecenter-to-center distances (the spacing) between objects that werespaced originally as in FIG. 8, but after the objects have been passedthrough the reformatting zone of the microfluidic system of FIG. 1, inaccordance with aspects of the present disclosure.

FIG. 10 is a schematic view of another exemplary microfluidic system forarranging objects, in accordance with aspects of the present disclosure.

FIG. 11 is a view of an exemplary rectangular geometry for thereformatting zone of the microfluidic system of FIG. 1, with thereformatting zone having a uniform depth (and no object-excludingregion), in accordance with aspects of the present disclosure.

FIG. 12 is a view of another exemplary rectangular geometry for thereformatting zone of the microfluidic system of FIG. 1, with thereformatting zone having object-excluding regions of reduced depth toreceive carrier fluid while restricting lateral travel of the objects,in accordance with aspects of the present disclosure.

FIG. 13 is a view of an exemplary rounded geometry for the reformattingzone of the microfluidic system of FIG. 1, with the reformatting zonehaving a uniform depth (and no object-excluding region), in accordancewith aspects of the present disclosure.

FIG. 14 is a view of another exemplary rounded geometry for thereformatting zone of the microfluidic system of FIG. 1, with thereformatting zone having object-excluding regions of reduced depth toreceive carrier fluid while restricting lateral travel of the objects,in accordance with aspects of the present disclosure.

FIG. 15 is a view of an exemplary tapered geometry for the reformattingzone of the microfluidic system of FIG. 1, with the reformatting zonehaving a uniform depth (and no object-excluding region), in accordancewith aspects of the present disclosure.

FIG. 16 is a view of another exemplary tapered geometry for thereformatting zone of the microfluidic system of FIG. 1, with thereformatting zone having object-excluding regions of reduced depth toreceive carrier fluid while restricting lateral travel of the objects,in accordance with aspects of the present disclosure.

FIG. 17 is a view of an exemplary branched geometry for the reformattingzone of the microfluidic system of FIG. 1, with the reformatting zonehaving a primary channel and a pair of lateral by-pass channels thatbranch from the primary channel upstream and rejoin the primary channeldownstream, in accordance with aspects of the present disclosure.

FIG. 18 is a view of another exemplary branched geometry for thereformatting zone of the microfluidic system of FIG. 1, with thereformatting zone being similar to that of FIG. 17, except having aprimary channel that tapers in the reformatting zone to place objects insingle file, in accordance with aspects of the present disclosure.

FIG. 19 is a schematic view of an exemplary embodiment of the system ofFIG. 1 being used to form an emulsion, to enable next generationsequencing, in accordance with aspects of the present disclosure.

FIG. 20 is a fragmentary view of a bottom section of an embodiment of achannel-defining microfluidic device of the system of FIG. 19, takenaround the reformatting zone of the device, in accordance with aspectsof the present disclosure.

DETAILED DESCRIPTION

The present disclosure provides microfluidic systems and methods forarranging a set of objects. In an exemplary method, the set of objectsmay be transported in carrier fluid along a microfluidic channelstructure having a reformatting zone including an object-accessibleregion and at least one object-excluding region. A portion of thecarrier fluid may be moved from the object-accessible region to the atleast one object-excluding region in an upstream section of thereformatting zone, to reduce a spacing of objects of the set. Theportion of the carrier fluid may be directed into the object-accessibleregion from the at least one object-excluding region in a downstreamsection of the reformatting zone, to increase a spacing of objects ofthe set. The steps of moving and directing in combination may increasethe spacing between objects disproportionately for a subset of theobjects that are closest to one another. The method may produce a moreuniform spacing of objects and/or a lower incidence of objects in closeproximity to one another.

The present disclosure provides an exemplary method of altering thespacing of a stream of objects. The stream of objects may be transportedin carrier fluid along a microfluidic channel structure defining anobject-excluding flow path for the objects and having a reformattingzone including an upstream section and a downstream section. A portionof the carrier fluid may be moved out of the flow path in the upstreamsection such that objects within the stream move closer to one another.The portion of the carrier fluid may be directed into the flow path inthe downstream section to increase a distance between objects within thestream. The stream may have a more uniform spacing of the objectsdownstream of the downstream section compared to upstream of theupstream section, and/or a lower incidence of objects in close proximityto one another downstream relative to upstream of the reformatting zone.

The general concept is to have a stream of carrier fluid (e.g., a liquidstream) flowing in a microfluidic channel and carrying objects (e.g.droplets, particles, etc.). The objects may have a size in the samerange as a transverse dimension of the microfluidic channel (e.g., 1-500micrometers). Various exemplary microfluidic designs are disclosed whichalter the spacing between these objects within the channel. An exemplarypurpose is to reduce the likelihood of two adjacent objects traveling inclose proximity to one another in the stream of carrier fluid, and/or torender the spacing of the objects more uniform within the stream ofcarrier fluid. Close proximity may be defined as a percentage of theaverage spacing between objects, such as less than 100%, 80% 60%, 50%,40%, 30%, 25%, 20%, or 10% of the average spacing.

Altering the spacing of the objects could be beneficial for variousapplications. For example, the application could be spacing of afluorescent readout from the objects. Other applications involvepartitioning a liquid stream carrying the objects. For example, if theliquid stream is to be partitioned into a plurality of isolated(separate) fluid volumes, there is a certain probability (typically, aPoisson distribution) that a given number of objects (i.e., 0, 1, 2, 3,etc.) will end up in the same fluid volume. By altering the spacing ofthe objects, and particularly objects that are in close proximity to oneanother, the probability of having two objects very close to one anothercan be reduced. As a result, the altered spacing creates a lowerprobability of having two or more objects within the same fluid volume.Essentially, this may overcome an underlying Poisson probability.

Exemplary microfluidic channel structures disclosed herein may utilize asimilar concept. In these designs, a portion of the carrier fluid may bedrained away from the objects by moving the portion to one or moreobject-excluding regions of the channel structure. This movement ofcarrier fluid concentrates objects within an object-accessible region ofthe channel structure. Objects that are farther apart get closer to oneanother. Objects that already are close to one another may get evencloser. However, since the objects have a finite size, there is aphysical limit to how closely the centers of the objects can approachone another before further approach is mutually obstructed. Accordingly,the objects may be concentrated differentially, with a subset of theobjects that are closest together being concentrated less than objectsthat are farther apart. The portion of carrier fluid may be reintroducedinto the object-accessible region of the channel structure, therebydiluting objects within the channel to increase the spacing thereof. Theobjects may be diluted equally. Therefore, the spacing between a subsetof the objects, namely, each adjacent pair of objects that exhibitedmutual obstruction during concentration, may be increaseddisproportionately by concentration and dilution. Furthermore,concentrating and diluting objects may create a larger minimum spacingbetween the objects. The minimum spacing may be determined by the sizeof the objects and the amount of carrier fluid that was drained from andreintroduced into the channel. The minimum spacing between objectsbefore concentration may be about the same as the diameter of theobjects. The minimum spacing after dilution may be at least about 125%,150%, 175%, 200%, or 250%, among others, of the diameter of the objects.The minimum spacing between objects may be increased at least 25%, 50%,75%, 100%, or 150%, among others, by concentration and dilution.

Further aspects of the present disclosure are described in the followingsections: (I) definitions, (II) overview of microfluidic systems andmethods for arranging objects, (III) reformatting zones, and (IV)examples.

I. Definitions

Technical terms used in this disclosure have meanings that are commonlyrecognized by those skilled in the art. However, the following terms maybe further defined as described below.

Object—any entity having a diameter of less than one millimeter. Theobjects of a set or object stream may have any suitable diameter, suchless than 500, 200, or 100 micrometers and/or greater than 1, 2, 5, 10,or 20 micrometers, among others. In exemplary embodiments, the objectsmay have a width of 1-500 micrometers, 5-500 micrometers, 10-200micrometers, or the like. The objects of a set or object stream may beabout the same size. For example, the objects may have a standarddeviation of their respective diameters that is less than about 40%,30%, 20%, 15%, or 10% of the mean of the diameters. A “stream” ofobjects may be a substantially single-file arrangement of the objects,whether or not the objects are exactly aligned with one another along aflow axis. Accordingly, the objects of an object stream may be offsetsomewhat from one another laterally, such as offset from a flow axis byless than about 75%, 50%, or 25% of the diameter of the objects. Anobject stream interchangeably may be described as a line, train,succession, or series of objects.

The objects may have any suitable shape. The objects of a set or linemay or may not be rounded and/or elongated. The present disclosureutilizes spherical objects to illustrate the systems and methods, andthe term “diameter” to describe the width of the objects. However, theterm “diameter” is intended to mean width (e.g., an average width) forany shape of object.

The objects may be composed of matter having any suitable state, such assolid, liquid, gas, or a combination thereof. However, the objects maybe predominantly solid, predominantly liquid, and/or predominantly acombination of solid and liquid, in some embodiments. The objects may besubstantially incompressible and/or rigid when subjected to themicrofluidic manipulations disclosed herein. Exemplary objects includeparticles, droplets, and the like. The particles may be biological cellsor beads, among others. The droplets may be composed at leastpredominantly of liquid.

Spacing—a distance between an adjacent pair of objects, a set ofindividual distances between adjacent objects of a group, object stream,or set, or an average distance between adjacent objects of a group,object stream, or set. The distance (a center-to-center spacing) betweenan adjacent pair of objects may be measured from the center of one ofthe objects to the center of the other object. This distance may bedescribed as a separation or separation distance between the objects,whether or not there is any space between the objects.

Microfluidic—involving fluid manipulation on a sub-millimeter scale. Forexample, a microfluidic channel may have a characteristic dimension,such as a width or depth, of less than one millimeter. Microfluidicsystems, and channels thereof, may produce and guide a laminar flow offluid, and/or may operate with a Reynolds number of less than about 500,200, 100, or 50, among others.

Carrier fluid—a fluid in which objects are transported in a microfluidicsystem, channel structure, and/or channel. The carrier fluid may beliquid. In some embodiments, the carrier fluid may be aqueous (or may benon-aqueous). The carrier fluid may be immiscible with liquid of theobjects, if the objects include liquid. If the objects are solid, theobjects may be substantially insoluble in the carrier fluid. In someembodiments, the carrier fluid may comprise oil, and the objects may beaqueous droplets, or the carrier fluid may be aqueous, and the objectsmay be droplets comprising oil, among others.

Channel—an elongated, fluid-guiding structure. A channel may be enclosedradially along most or all of its longitudinal extent.

Channel structure—a fluid-guiding structure including a single channelor two or more channels that communicate with one another. The channelstructure may be unbranched or branched. Each of the channels may be amicrofluidic channel. The channel structure may be configured to guidefluid from an inlet to an outlet, without any substantial addition orloss of fluid. The channel structure may have a cross-sectional area forfluid flow at each position between the inlet and the outlet. Thecross-sectional area may be defined at each position by a singlechannel, or collectively by two or more channels configured to carryfluid in parallel.

II. Overview of Microfluidic Systems and Methods for Arranging Objects

This section provides an overview of exemplary microfluidic systems andmethods for arranging objects; see FIGS. 1-10.

FIG. 1 shows a schematic view of an exemplary microfluidic system 50 forarranging objects, and particularly for reformatting a stream 52 ofobjects 54 to space the objects more uniformly. The system includes achannel structure 56 having an inlet 58 (e.g., a single inlet), anoutlet 60 (e.g., a single outlet), and a reformatting zone 62 located onan object-accessible flow path 63 that connects the inlet to the outlet.The channel structure may have only one channel 64 extending between theinlet and outlet and forming the reformatting zone, as in the depictedembodiment. In other embodiments, channel structure 56 may branchupstream and merge downstream to create the reformatting zone (seeSection II).

Channel structure 56 may be configured to direct a stream 66 of carrierfluid 68 transporting objects 54 from inlet 58 to outlet 60, with theobjects following, and substantially restricted to, flow path 63. Theobjects may be arranged in object stream 52, which may be straight,bent, curved, or the like. In other embodiments described below, theobjects may be placed into single file by the reformatting zone, andthus the objects may enter the reformatting zone laterally spread out.

Reformatting zone 62 may have a fixed geometry that passively (e.g.,without valves, feedback, electrical signals, etc.) adjusts the spacingbetween at least a subset of the objects, to make the spacing lessstochastic (such as to make the spacing less of a Poisson distribution).For example, in the depicted embodiment, objects 54 located upstream ofreformatting zone 62 in an inflow region 70 of the channel structurehave a more variable spacing than objects 54 located downstream ofreformatting zone 62 in an outflow region 72 of the channel structure.More particularly, inflow region 70 contains two unseparated pairs 74 a,74 b of objects, while all of objects 54 in outflow region 72 are wellseparated from one another. As described further below, the reformattingzone may be configured to disproportionately increase the relativeand/or absolute spacing between adjacent objects that are spaced fromone another by less than a threshold distance.

Reformatting zone 62 may be configured to concentrate and diluteobjects, to produce a change in the spacing between the objects. Thezone may have an object-accessible region 76 (e.g., the part of flowpath 63 passing through zone 62) in which objects 54 can travel, and oneor more object-excluding regions 77 that selectively and/orsubstantially exclusively permit entry of carrier fluid 68 relative toobjects 54. Object-excluding regions 77 may be configured to exclude amajority of the objects from entering the region, such as more thanabout 70%, 80%, 90%, 95,%, 98% or 99% of the objects, among others.

Reformatting zone 62 may have a concentration section 78,interchangeably called an upstream section, in which a portion ofcarrier fluid 68 is moved from flow path 63, indicated by arrows at 80.The portion of carrier fluid is moved from object-accessible region 76to object-excluding regions 77. The portion of carrier fluid may bemoved into one or more wings 82 (i.e., one or more lateral areas) ofchannel 64 (and/or into one or more by-pass channels) that branch fromthe channel and are separate from one another.

Reformatting zone 62 also may have a dilution section 84,interchangeably called a downstream section or re-entry section, inwhich the portion of carrier fluid 68 may be reintroduced into flow path63 (and thus into object-accessible region 76) from object-excludingregions 77 (e.g., from one or more wings 82), indicated by arrows at 86.The velocity of objects 54 may (or may not) decrease in concentrationsection 78, and/or carrier fluid may be drained from theobject-accessible region of the reformatting zone, which may causeobjects 54 to be concentrated locally. The local spacing of objects 54in the reformatting zone may decrease, relative to their spacing ininflow region 70. The velocity of objects 54 may increase, and/orcarrier fluid 68 may be reintroduced into object-accessible region 76 indilution section 84, which may cause the spacing between objects 54,such as the spacing for each adjacent pair 74 a, 74 b, to be increasedfrom the more concentrated configuration in the reformatting zone.

Reformatting zone 62 may have any suitable length. For example, thelength may be at least about 2, 3, 4, 5, 10, or 20 object diameters,among others.

FIG. 2 shows an exemplary geometry for channel structure 56 upstream(and/or downstream) of reformatting zone 62. The channel structure maybe formed by a channel-defining device 87 having a plurality of layers,such as layers 88, 90, bonded to one another and defining a plane 92.Channel 64, in inflow region 70, and/or outflow region 72, may be sizedin correspondence with objects 54 (see FIGS. 1 and 2). The channel mayhave a depth 94 (measured orthogonal to plane 92) and a width 96(measured parallel to plane 92) that are about the same as one another,such as less than about 100%, 75%, 50%, or 25% different. The size ofchannel 64 in inflow region 70 and outflow region 72 may be the same, ormay be different. For example, the outflow region may have a smallercross-sectional area, depth, and/or width than the inflow region (alsosee below). The depth and/or width of the outflow region and/or inflowregion may be only somewhat larger than the diameter of objects 54, suchas less than about 100%, 75%, 50%, or 25% larger, among others, toreduce lateral migration of the objects out of alignment with oneanother.

FIG. 3 shows an exemplary geometry for channel structure 56 withinreformatting zone 62. Channel 64 may (or may not) have an expandedregion 98 (also see FIG. 1) at which a width 100 of the channelincreases relative to width 96 of inflow region 70 and/or outflow region72. Whether or not the channel has an expanded region, a depth 104 ofobject-excluding region(s) 77 (e.g., depth 104 of each wing 82) may beless than a depth 106 of object-accessible region 76, to form a shelf onone or both sides of object-accessible region 76. Depth 104 of eachobject-excluding region 77 may be less than the diameter of objects 54,to substantially exclude objects 54 from the object-excluding region.Accordingly, each object-excluding region 77 may be configured to permitselective movement of only carrier fluid 68, and not objects 54, fromobject-accessible region 76, making the process of draining carrierfluid from around objects 54 more efficient. Each object-excludingregion 77 may have a substantially uniform depth, or the depth may varystepwise or via a taper, among others, within the object-excludingregion. Object-excluding regions 77 of FIGS. 1-3 and elsewhere hereinare shown as being symmetrical, with object-accessible region 76 locatedbetween, contiguous with, and separating, a pair of object-excludingregions. However, in other embodiments, only a single object-excludingregion 77 may be located on only one side of object-accessible region76, the object-excluding regions may be asymmetrically arranged, orthree or more object-excluding regions may be present.

Object-accessible region 76 may be formed adjacent each object-excludingregion 77 at least in part by a groove 108. The object-accessible regionmay include laterally unbounded space above the groove, as indicated bya pair of dashed boundaries extending upward from the walls of thegroove between wings 82.

The amount of increase and decrease in cross-sectional area of thechannel structure created by expanded region 98 (and/or by one or moreby-pass channels) may directly related to the fraction of carrier fluidthat is drained from the object-accessible flow path and reintroduced tothe flow path. Accordingly, a larger increase and decrease incross-sectional area may provide a greater deceleration and accelerationof objects, and a larger effect on object spacing. The amount ofincrease in cross-sectional area may set a threshold separation distancebetween objects, below which the relative or absolute spacing withinobject pairs is increased disproportionately by the reformatting zone.With a smaller increase in cross-sectional area, the spacing betweenonly closely paired objects may be affected disproportionately, whilewith a larger increase in cross-sectional area, the spacing within agreater percentage of the object pairs may be altereddisproportionately.

FIG. 1 shows other exemplary aspects of system 50. One or more sourcesof positive/negative pressure, such as at least one pump 110, may beoperatively connected to channel structure 56 upstream of inlet 58and/or downstream of outlet 60. The pump(s) creates a pressuredifferential to move fluid within the channel structure. Each pressuresource may be any device or mechanism configured to drive flow ofcarrier fluid 68 and objects 54 from inlet 58 to outlet 60. Each pumpmay, for example, apply positive pressure upstream of inlet 58,indicated at 112, to push carrier fluid, or may apply negative pressure(suction) downstream of outlet 60, indicated at 114, to pull carrierfluid. Exemplary pumps may include syringe pumps, peristaltic pumps,diaphragm pumps, piston pumps, and the like.

A source 116 of objects 54 and carrier fluid 68 may be connected tochannel structure 56. Source 116 may be connected removably orintegrally, among others. Objects 54 may be placed into single file asthe objects enter channel structure 56. For example, the objects maytravel through a tapered alignment region 118 leading to inlet 58. Inother embodiments, alignment region 118 may be provided by reformattingzone 62 (see below).

System 50 may have a partitioning structure 120 located at or downstreamof outlet 60. The partitioning structure may be configured to dividecarrier fluid stream 66 into a plurality of isolated volumes 122(interchangeably called partitions), optionally of equal size. The sizeof the volumes may be selected such that a majority of the volumescontain either no object 54 or only one object 54. Passing carrier fluidstream 66 through reformatting zone 62 before partitioning can makepartitioning less random, by decreasing the percentage of volumescontaining two or more objects 54 and increasing the percentage ofvolumes 122 containing only one object

Partitioning structure 120 may have any suitable mechanism of operation.The partitioning structure may be a dispenser that dispenses volumes 122into separate containers or as an aerosol. In other embodiments, thepartitioning structure may form volumes 122 encapsulated by a continuousliquid phase. Accordingly, the partitioning structure may be a dropletgenerator. Exemplary droplet generators form droplets by flow focusing,shearing, co-flow, a confinement gradient, etc.

In some embodiments, system 50 may include a detector 124 operativelylocated downstream of reformatting zone 62. Detector 124 may beconfigured to detect a signal from a detection zone 126 of channel 64 asobjects 54 pass therethrough. The reformatting zone increases theseparation between closely paired objects and thus may reduce theincidence of signal overlap between detected waveforms corresponding tothe objects. The detector may, for example, be configured to detectlight from detection zone 126. In some embodiments, a light source 128may be configured to irradiate the detection zone, such as to producephotoluminescence from the objects. The light source may provide epi- ortrans-illumination of the detection zone. Partitioning structure 120 mayor may not be omitted from embodiments including detector 124.

FIGS. 4-7 show part of channel structure 56 of system 50 of FIG. 1 inthe presence of a pair of objects 54 (“A” and “B”). The upstream,center-to-center distance, spacing S1, between the objects in inflowregion 70 is different in each figure to illustrate how reformattingzone 62 may operate on objects of different initial spacing. The samepair of objects 54 in each figure is shown as dashed inside reformattingzone 62, and in dash-dot-dot outline in outflow region 72 after passingthrough the reformatting zone. The spacing for the pair of objects ateach of the three positions along the flow path is labeled as S1, S2,and S3, respectively. The changes in spacing shown in FIGS. 4-7 areexemplary; other channel geometries may produce larger or smallerchanges. Furthermore, the reversible changes in separation shown inFIGS. 6 and 7 for S1 greater than the threshold distance may not applyin some case, such as if the cross-sectional areas of inflow region 70and outflow region 72 are different.

FIG. 4 shows a configuration in which S1 is approximately equal to thediameter of the pair of objects. In other words, the objects are veryclose to one another before entering reformatting zone 62. Accordingly,although moving carrier fluid into object-excluding regions 77 inconcentration section 78 urges the objects toward one another, theobjects cannot move substantially closer to one another, and S1substantially equals S2, because the objects mutually obstruct oneanother. However, the dilution produced by dilution section 84 causesthe objects to move apart from one another, such that S3 is greater thanS1 (and S2).

FIG. 5 shows a configuration in which S1 is greater than in FIG. 4, butthe two objects are still relatively close (e.g., S1 may be less thantwo object diameters). Moving carrier fluid into object-excludingregions 77 in concentration section 78 may urge the objects closertogether until the objects mutually obstruct further movement toward oneanother. At this point, S2 is substantially equal to the diameter of theobjects (i.e., S1), and no further movement toward each other ispermitted without object deformation. As in FIG. 4, the dilutionproduced by dilution section 84 causes the objects to move apart fromone another, such that S3 is greater than S1 (and S2). S3 in FIGS. 4 and5 may be the same, since S2 is the same, even though S1 is different.Accordingly, each pair of objects having less than a threshold spacingfor S1 may have the same spacing S3 as one another after passing throughthe reformatting zone. The threshold spacing may be determined by thediameter of the objects, and a ratio of the amount of concentration andthe amount of dilution produced by the reformatting zone.

FIGS. 6 and 7 show configurations in which S1 is greater than in FIG. 5(and greater than the threshold spacing). More particularly, S1 is largeenough that moving carrier fluid into object-excluding regions 77 inconcentration section 78 urges the objects closer together but not closeenough for the objects to obstruct movement toward one another.Accordingly, the dilution produced by dilution section 84 causes theobjects to move apart from one another to their original spacing (i.e.,S1 equals S3). At the threshold spacing (e.g., intermediate S1 of FIGS.5 and 6), the objects may move to the closest approach of FIGS. 4 and 5but still may return to their original spacing. Any adjacent objectshaving less than the threshold spacing for S1 leave the reformattingzone with substantially the same spacing S3, which is the reformattedminimum spacing between objects.

FIG. 8 shows a histogram presenting an exemplary distribution of thespacing between objects 54 upstream of reformatting zone 62. Objectsthat are relatively closer together (e.g., represented by the bar on theleft) may be undesirable for applications in which the objects should bewell singulated.

FIG. 9 shows a histogram presenting an exemplary change in thedistribution of FIG. 8 produced by passing the objects throughreformatting zone 62 of system 50. In FIG. 9, the threshold spacing forreformatting zone 62 (see FIGS. 5 and 6) is between two and three.Accordingly, the object pairs that were represented by the bar on theleft (in FIG. 8) have been spaced farther from one another and are nowrepresented by the middle bar of the histogram. However, the spacing ofthe rest of the object pairs may not have changed significantly.Accordingly, the spacing of the objects has been adjusted to increasethe average distance between objects, by selectively (and/ordisproportionately) increasing the spacing between a subset of theobjects that are closest to one another.

FIG. 10 shows another exemplary microfluidic system 50′ for arrangingobjects 54. System 50′ may have any suitable combination of featuresdescribed above for system 50 (see FIG. 1), such as a pump 110 to driveflow of carrier fluid 68 and objects 54, a partitioning structure 120, adetector 124 in communication with a detection zone 126, and the like.

The system also may include a channel structure 56 forming areformatting zone 62. The reformatting zone may be created by a singlechannel 64 or by two or more channels of the channel structure, asdescribed below in Section III. The reformatting zone may include anobject-accessible region 76 and one or more object-excluding regions 77,which may be of different depth from one another, as described above forsystem 50 (see FIG. 3). Moreover, the reformatting zone may have aconcentration section 78 in which a portion of carrier fluid 68 is movedfrom object-accessible region 76 to object-excluding regions 77,indicated by arrows at 80. Reformatting zone 62 also may have a dilutionsection 84 in which the portion of carrier fluid is reintroduced toobject-accessible region 76 from object-excluding regions 77, indicatedby arrows at 86.

However, reformatting zone 62 of system 50′ may not be formed by anexpanded region of channel 64 (compare with expanded region 98 of FIG.1). Instead, channel 64, at the upstream end of reformatting zone 62,may be much wider than the diameter of objects 54 (e.g., more than 2, 3,4, or 5 times the diameter of the objects), and may narrow toward thedownstream end of reformatting zone 62. The objects may enter thereformatting zone spread out laterally from one another in a disorderedarrangement (and not aligned). Objects 54 may be aligned with oneanother and placed into single file by tapered alignment region 118 ofreformatting zone 62 (namely, object-accessible region 76 thereof) asthe objects travel downstream through the reformatting zone. Alignmentregion 118 thus may be contiguous with each object-excluding region 77(and one or more wings 82). The cross-sectional area for fluid flow maytaper from the upstream end to the downstream end of the reformattingzone. Accordingly, in contrast to system 50, carrier fluid 68 may not bedecelerated as it enters the reformatting zone.

III. Reformatting Zones

This section describes other exemplary channel geometries forreformatting zone 62 of system 50 and/or 50′; see FIGS. 11-18 (also seeFIGS. 1-10). The outline convention for each pair of objects “A” and “B”in FIGS. 11-18 is as described above in Section II.

FIGS. 11-16 show three different geometries for reformatting zone 62,namely rectangular (FIGS. 11 and 12), round (FIGS. 13 and 14), andtapered (FIGS. 15 and 16). For each type of geometry, two embodimentsare illustrated: one having uniform channel depth (FIGS. 11, 13, and15), and the other having a deeper object-accessible region 76 andshallower object-excluding regions 77.

FIG. 11 shows an exemplary rectangular geometry for reformatting zone 62of system 50. The zone has an expanded region 98 to create upstreamdeceleration and downstream acceleration within the zone. However, thedepth of reformatting zone 62 is uniform, such that carrier fluid 68 isnot drained away from objects 54 efficiently.

FIG. 12 shows another exemplary rectangular geometry for reformattingzone 62 of system 50. The perimeter of the zone has the same shape as inFIG. 11. However, the depth of zone 62 varies, as described above forsystem 50 of FIG. 1, to create object-accessible region 76 andobject-excluding regions 77. The presence of object-excluding regions 77allows the geometry of FIG. 12 to drain carrier fluid away from objects54 much more efficiently than in FIG. 11, to produce a much greaterconcentration and dilution of objects by the zone. FIGS. 13 and 14 havethe same general relation to one another as FIGS. 11 and 12, as do FIGS.15 and 16.

FIG. 17 shows an exemplary branched geometry 130 for reformatting zone62 of system 50. The zone has a primary channel 132 and one or morelateral by-pass channels 134. Each by-pass channel branches from primarychannel 132 and rejoins the primary channel downstream. The primarychannel forms object-accessible region 76, and the by-pass channels formobject-excluding regions 77. Carrier fluid 68 may enter by-pass channels134 in concentration section 78, to concentrate objects 54 in primarychannel 132. Carrier fluid 68 may be reintroduced in dilution section 84to dilute the objects. An inlet 136 of each by-pass channel 134, formedat the downstream end of an inflow channel 138, may be configured toexclude objects 54. For example, the inlet may have a width and/or adepth that is less than the diameter of the objects, and/or the inletmay include one or more pillars or other barriers to object entry. Anoutflow channel 140 may extend downstream from reformatting zone 62.

FIG. 18 shows another exemplary branched geometry 130 for reformattingzone 62 of system 50. The geometry of the reformatting zone of FIG. 18is similar to that of FIG. 17 except that inflow channel 138 is muchwider than the diameter of objects 54 (as in system 50′ of FIG. 10).Also, primary channel 132 within zone 62 forms a tapered alignmentregion 118 (as in system 50′ of FIG. 10).

IV. EXAMPLES

The following examples describe selected aspects and embodiments ofmicrofluidic systems and methods for arranging objects. These aspectsand embodiments are intended for illustration and should not limit theentire scope of the present disclosure.

Example 1 System and Method for Next Generation Sequencing

This example describes an exemplary system 150 and method for generatingan emulsion 152 to enable next generation sequencing. The emulsion mayinclude isolated volumes (droplets 154) of carrier fluid 68 encapsulatedby an immiscible continuous phase liquid 156 (e.g., oil). Droplets 154may contain beads 158 (as objects 54) and biological cells 160 atpartial occupancy; see FIGS. 19 and 20. Beads 158 may carryoligonucleotides, which may be configured to function as primers and/orbarcodes.

System has a channel structure including bead channel 162, at least onecell channel 164, at least one bead-and-cell channel 166, one or moreoil channels 168, and a droplet channel 170. Bead channel 162 carriesbeads 158 through reformatting zone 62 to a junction with cell channel164. Cells 160 are introduced to a bead-containing stream 172 at thejunction to produce bead-and-cell-containing stream 174. Stream 174travels to a channel junction 176 at which the stream may be segmentedby at least one stream of continuous phase liquid 156 to generatedroplets 154.

The goal is to have as many droplets as possible containing only onebead and only one cell, while minimizing the number of dropletscontaining two or more beads. With a Poisson distribution of the beads(no reformatting zone 62), the percentage of droplets containing onebead is kept relatively low, to avoid an unacceptably high fraction ofdroplets with two beads. Zone 62 permits a greater percentage ofdroplets (e.g., ˜40%) to contain one bead, while fewer (e.g., ˜5%)contain two beads. The percentage with three beads may be negligible.

FIG. 20 shows a bottom section of a channel-defining microfluidic device178 of an embodiment of system 150. Reformatting zone 62 has a wideexpanded region 98 with a deep entry region 180 of uniform depth inwhich objects may accumulate. The expanded region becomes shallower toform a downstream portion of object-accessible region 76, and evenshallower still to form lateral object-excluding regions 77. Thedownstream portion of object-accessible region 76 tapers to createalignment region 118 for the objects. A pair of cell channels 164 a, 164b intersect the outlet of reformatting zone 62, to add biological cellsto the outflowing stream of carrier fluid and beads. A resulting streamof carrier fluid transporting beads and biological cells travels alongbead-and-cell channel 166.

Example 2 Selected Embodiments

This example describes selected embodiments of the present disclosurepresented as a series of indexed paragraphs.

Paragraph A1. A method of arranging objects, the method comprising: (a)transporting a set of objects in carrier fluid along a microfluidicchannel structure having a reformatting zone including anobject-accessible region and at least one object-excluding region; (b)moving a portion of the carrier fluid from the object-accessible regionto the object-excluding region(s) in an upstream section of thereformatting zone, to reduce a spacing of objects of the set; and (c)directing the portion of the carrier fluid into the object-accessibleregion from the object-excluding region(s) in a downstream section ofthe reformatting zone, to increase a spacing of objects of the set.

Paragraph A2. The method of paragraph A1, wherein at least one pair ofobjects of the set are in sufficiently close proximity to one another inthe reformatting zone that the objects of the pair mutually obstruct acloser approach to one another encouraged by the step of moving.

Paragraph A3. The method of paragraph A1 or A2, wherein a spacingbetween a subset of the objects of the set that are closest together isincreased disproportionately by the steps of moving and directing incombination.

Paragraph A4. The method of any of paragraphs A1 to A3, wherein objectsof the set leave the reformatting zone in single file.

Paragraph A5. The method of any of paragraphs A1 to A4, furthercomprising a step of arranging objects of the set in single file.

Paragraph A6. The method of paragraph A5, wherein objects of the set arearranged in single file by an alignment region of the channel structurethat does not overlap the upstream section.

Paragraph A7. The method of paragraph A5, wherein objects of the set arearranged in single file by an alignment region of the channel structurelocated in the upstream section.

Paragraph A8. The method of paragraph A6 or A7, wherein the alignmentregion tapers to a width that is less than twice an average diameter ofthe objects.

Paragraph A9. The method of any of paragraphs A1 to A8, wherein theobject-accessible region is deeper than each object-excluding region ofthe at least one objection-excluding region.

Paragraph A10. The method of paragraph A9, wherein the object-excludingregion has a depth that is less than an average diameter of the objectsof the set.

Paragraph A11. The method of paragraph A9 or A10, wherein theobject-accessible region and each object-excluding region of the atleast one object-excluding region are formed by a same channel of thechannel structure.

Paragraph A12. The method of paragraph A11, wherein eachobject-excluding region of the at least one object-excluding region iscontinuously contiguous with the object-accessible region between theupstream section and the downstream section.

Paragraph A13. The method of any of paragraphs A9 to A12, wherein theobject-accessible region includes an object-accessible groove, andwherein the at least one object-excluding region includes one or morewings located adjacent the object-accessible groove.

Paragraph A14. The method of any of paragraphs A9 to A13, wherein the atleast one object-excluding region includes a pair of object-excludingregions that are separated from one another by the object-accessibleregion, and optionally separated from one another only by theobject-accessible region.

Paragraph A15. The method of any one of paragraphs A1 to A14, whereinthe object-accessible region is formed by a primary channel of thechannel structure, and wherein the step of moving includes a step ofmoving at least part of the portion of the carrier fluid to one or moreby-pass channels defined by the channel structure.

Paragraph A16. The method of paragraph A15, wherein each by-pass channelbranches from the primary channel in the upstream section and mergeswith the primary channel in downstream section.

Paragraph A17. The method of paragraph A15 or A16, wherein the one ormore by-pass channels include a pair of by-pass channels, and whereinthe primary channel is located between the pair of by-pass channels.

Paragraph A18. The method of any one of paragraphs A15 to A17, whereineach by-pass channel has an inlet configured to prevent entry of objectsof the set into the by-pass channel.

Paragraph A19. The method of paragraph A18, wherein the inlet is sizedto prevent entry of objects of the set into the by-pass channel.

Paragraph A20. The method of any one of paragraphs A1 to A19, wherein aplurality of objects of the set travel through the upstream sectionduring the step of moving, and wherein only a subset of the plurality ofobjects move closer to an adjacent object of the set during the step ofmoving without being stopped by a periphery of the adjacent object.

Paragraph A21. The method of paragraph A20, wherein at least a pair ofthe plurality of objects do not move substantially closer to one anotherduring the step of moving due to mutual obstruction.

Paragraph A22. The method of any one of paragraphs A1 to A21, wherein aplurality of objects of the set travel through the downstream sectionduring the step of directing, wherein each object of the plurality ofobjects moves farther from each adjacent object of the plurality duringthe step of directing,

Paragraph A23. The method of any one of paragraphs A1 to A22, whereinthe steps of moving and directing, in combination, create substantiallythe same spacing for a subset of pairs of the objects that are closestto one another.

Paragraph A24. The method of paragraph A23, wherein objects of each ofthe pairs are spaced from one another by less than a threshold spacingbefore traveling through the formatting zone.

Paragraph A25. The method of any one of paragraphs A1 to A24, whereinthe carrier fluid is an aqueous carrier fluid.

Paragraph A26. The method of any one of paragraphs A1 to A25, whereinthe objects include liquid that is immiscible with the carrier fluid,and wherein, optionally, each of the objects is formed at leastpredominantly of liquid.

Paragraph A27. The method of any one of paragraphs A1 to A26, whereinthe objects are selected from the group consisting of beads, droplets,and biological cells.

Paragraph A28. The method of any one of paragraphs A1 to A27, furthercomprising a step of partitioning a stream including the carrier fluidand carrying objects of the set at a position downstream of thedownstream section to form isolated volumes.

Paragraph A29. The method of paragraph A28, wherein each isolated volumeof a majority of the isolated volumes contains none or only one of theobjects of the set.

Paragraph A30. The method of paragraph A28 or A29, wherein the step ofpartitioning includes a step of encapsulating the isolated volumes witha liquid that is immiscible with the carrier fluid.

Paragraph A31. The method of any one of paragraphs A28 to A30, whereinthe step of partitioning includes a step of forming droplets.

Paragraph A32. The method of any one of paragraphs A28 to A31, whereinthe objects are beads, further comprising a step of adding biologicalcells to the carrier fluid.

Paragraph A33. The method of paragraph A32, wherein each isolated volumeof a plurality of the isolated volumes contains only one of the beadsand only one biological cell.

Paragraph A34. The method of any one of paragraphs A1 to A33, furthercomprising a step of moving one or more of the objects of the setthrough a detection zone downstream of the downstream section, and astep of detecting a signal from the detection zone.

Paragraph A35. The method of paragraph A34, wherein the step ofdetecting a signal includes a step of detecting light.

Paragraph B1. A method of altering the spacing of a stream of objects,the method comprising: (a) transporting objects of the object stream incarrier fluid along an object- accessible flow path, the flow path beingdefined by a microfluidic channel structure and extending through areformatting zone including an upstream section and a downstreamsection; (b) moving a portion of the carrier fluid out of the flow pathin the upstream section such that objects within the object stream movecloser to one another; and (c) directing the portion of the carrierfluid into the flow path in the downstream section to increase adistance between objects within the object stream.

Paragraph B2. The method of paragraph B1, wherein the object stream hasa more uniform spacing of objects downstream of the downstream sectioncompared to upstream of the upstream section, and/or wherein a distancebetween object pairs of the object stream that are closest togetherupstream of the upstream section is increased disproportionately by thesteps of moving and directing in combination.

Paragraph B3. The method of paragraph B1 or paragraph B2, wherein thechannel structure includes an object-accessible groove and at least oneobject-excluding wing located adjacent the groove.

Paragraph B4. The method of paragraph B3, wherein the step of movingincludes a step of moving the portion of the carrier fluid from a deeperregion to at least one shallower region of the channel structure.

Paragraph B5. The method of paragraph B4, wherein the step of movingincludes a step of moving the portion of the carrier fluid to a pair ofshallower regions of the channel structure that are separated from oneanother by a deeper, object-accessible groove.

Paragraph B6. The method of paragraph B4 or B5, wherein the step ofmoving includes a step of moving the portion of the carrier fluid to atleast one shallower region having a depth that is less than a diameterof the objects, such that a majority of objects of the object stream areexcluded from the at least one shallower region.

Paragraph B7. The method of any one of paragraphs B1 to B6, wherein thestep of moving includes a step of moving the portion of the carrierfluid from a primary channel to one or more by-pass channels defined bythe channel structure.

Paragraph B8. The method of paragraph B7, wherein the channel structurehas a primary channel, and wherein each by-pass channel branches fromthe primary channel in the upstream section and merges with the primarychannel in the downstream section.

Paragraph B9. The method of paragraph B8, wherein the one or moreby-pass channels include a pair of by-pass channels.

Paragraph B10. The method of any one of paragraphs B7 to B9, whereineach by-pass channel has an inlet configured to prevent entry of objectsof the object stream into the by-pass channel.

Paragraph B11. The method of paragraph B10, wherein the inlet is sizedto prevent entry of objects of the object stream into the by-passchannel.

Paragraph B12. The method of any of paragraphs B1 to B11, wherein aplurality of the objects of the object stream travel through theupstream section during the step of moving, and wherein only a subset ofthe plurality of objects move closer to an adjacent object of the objectstream during the step of moving until closer approach is obstructed bya periphery of the adjacent object.

Paragraph B13. The method of any of paragraphs B1 to B12, wherein atleast a pair of the plurality of objects do not move substantiallycloser to one another during the step of moving due to mutualobstruction.

Paragraph B14. The method of any one of paragraphs B1 to B13, wherein aplurality of the objects travel through the downstream section duringthe step of directing, and wherein each object of the plurality ofobjects moves farther from each adjacent object of the object streamduring the step of directing.

Paragraph B15. The method of any one of paragraphs B1 to B14, whereinthe carrier fluid is an aqueous carrier fluid.

Paragraph B16. The method of any one of paragraphs B1 to B15, whereinthe objects are formed at least predominantly of liquid that isimmiscible with the carrier fluid.

Paragraph B17. The method of any one of paragraphs B1 to B16, whereinthe objects are selected from the group consisting of beads, droplets,and biological cells.

Paragraph B18. The method of any one of paragraphs B1 to B17, furthercomprising a step of forming partitions from a stream including thecarrier fluid and carrying a plurality of the objects at a position ofthe channel structure downstream of the downstream section.

Paragraph B19. The method of paragraph B18, wherein each partition of amajority of the partitions contains none or only one of the objects.

Paragraph B20. The method of paragraph B18 or B19, wherein the step offorming partitions includes a step of encapsulating volumes of thestream with an immiscible liquid.

Paragraph B21. The method of any one of paragraphs B18 to B20, whereinthe step of forming partitions includes a step of forming droplets.

Paragraph B22. The method of any one of paragraphs B18 to B21, whereinthe objects are beads, further comprising a step of adding biologicalcells to the carrier fluid.

Paragraph B23. The method of paragraph B22, wherein each partition of aplurality of the partitions contains only one of the beads and only onebiological cell.

Paragraph B24. The method of any one of paragraphs B1 to B23, furthercomprising a step of passing one or more of the objects through adetection zone downstream of the downstream section, and a step ofdetecting a signal from the detection zone.

Paragraph B25. The method of paragraph B24, wherein the step ofdetecting a signal includes a step of detecting light.

Paragraph C1. A method of altering the spacing of a stream of objects,the method comprising: (a) transporting objects of the object stream incarrier fluid along a microfluidic channel structure having adeceleration region upstream of an acceleration region; (b) slowing downobjects within the object stream at the deceleration region such that atleast a subset of such objects are moved closer to one another; and (c)speeding up objects of the object stream at the acceleration region toincrease a distance between such objects; wherein the object stream hasa lower incidence of closely-spaced pairs of the objects downstream ofthe acceleration region compared to upstream of the deceleration region.

Paragraph C2. The method of paragraph C1, further comprising any one orcombination of the limitations from paragraphs A1 to A35 and B1 to B25.

Paragraph D1. A method of altering the spacing of a stream of objects,the method comprising: (a) transporting the objects of the object streamin carrier fluid along a microfluidic channel structure having an inflowregion, an outflow region, and an expanded region extending from theinflow region to the outflow region, wherein the expanded region has agreater cross-sectional area for fluid flow than the inflow region andthe outflow region; (b) moving objects of the object stream from theinflow region to the expanded region such that at least a subset of suchobjects are moved closer to one another; and (c) passing objects of theobject stream from the expanded region to the outflow region to increasea distance between such objects.

Paragraph E1. A system for arranging a set of objects, comprising: (a) amicrofluidic channel structure having a reformatting zone including anobject-accessible region and at least one object-excluding region; and(b) at least one source of positive/negative pressure operativelyconnected to the channel structure and configured to form a stream ofcarrier fluid transporting objects of the set in the channel structure;wherein the channel structure is configured such that a portion of thecarrier fluid is moved from the object-accessible region to the at leastone object-excluding region in an upstream section of the reformattingzone, to reduce a spacing between objects of the set, and such that theportion of the carrier fluid is directed into the object-accessibleregion from the at least one object-excluding region in a downstreamsection of the reformatting zone, to increase a spacing between objectsof the set.

Paragraph E2. The system of paragraph E1, further comprising any one orcombination of the limitations from paragraphs A1 to A35, B1 to B25, C1to C2, and D1.

Paragraph F1. A system for altering the spacing of a stream of objects,comprising: (a) a microfluidic channel structure having an inflowregion, an outflow region, and an expanded region extending from theinflow region to the outflow region, wherein the expanded region has agreater cross-sectional area for fluid flow than the inflow region andthe outflow region; (b) a source of carrier fluid and objects incommunication with the inflow region of the channel structure; and (c)at least one source of positive/negative pressure operatively connectedto the channel structure and configured to form a stream of carrierfluid transporting a stream of objects, such that objects of the objectstream pass from the inflow region to the expanded region and at least asubset of such objects are moved closer to one another, such thatobjects of the object stream are directed from the expanded region tothe outflow region to increase a distance between such objects, and suchthat the object stream has a more uniform spacing of the objectsdownstream of the expanded region compared to upstream of the expandedregion.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.Further, ordinal indicators, such as first, second, or third, foridentified elements are used to distinguish between the elements, and donot indicate a particular position or order of such elements, unlessotherwise specifically stated. Finally, the present disclosureincorporates material by reference. If any ambiguity or conflict in themeaning of a term results from this incorporation by reference, theliteral contents of the application govern construction of the term.

We claim:
 1. A method of arranging objects, the method comprising:transporting a set of objects in carrier fluid along a microfluidicchannel structure having a reformatting zone including anobject-accessible region and at least one object-excluding region;moving a portion of the carrier fluid from the object-accessible regionto the at least one object-excluding region in an upstream section ofthe reformatting zone, to reduce a spacing of objects of the set in theupstream section; and directing the portion of the carrier fluid intothe object-accessible region from the at least one object-excludingregion in a downstream section of the reformatting zone, to increase aspacing of objects of the set in, and/or downstream of, the downstreamsection.
 2. The method of claim 1, wherein at least one pair of objectsof the set are in sufficiently close proximity to one another in thereformatting zone that the objects of the at least one pair mutuallyobstruct a closer approach to one another encouraged by the step ofmoving.
 3. The method of claim 1, wherein a spacing between a subset ofthe objects of the set that are closest together is increaseddisproportionately by the steps of moving and directing in combination.4. The method of claim 1, wherein objects of the set leave thereformatting zone in single file.
 5. The method of claim 1, wherein thereformatting zone has an outlet, further comprising arranging objects ofthe set in single file in the channel structure upstream of the outletof the reformatting zone.
 6. The method of claim 5, wherein objects ofthe set are arranged in single file by an alignment region of thechannel structure located in the upstream section.
 7. The method ofclaim 5, wherein objects of the set are arranged in single file by analignment region of the channel structure that tapers from the upstreamsection to the downstream section of the reformatting zone.
 8. Themethod of claim 7, wherein the alignment region tapers to a width thatis less than twice an average diameter of the objects.
 9. The method ofclaim 1, wherein the object-accessible region is deeper than eachobject-excluding region of the at least one object-excluding region. 10.The method of claim 9, wherein the at least one object-excluding regionhas a depth that is less than an average diameter of the objects of theset.
 11. The method of claim 9, wherein the object-accessible region andeach object-excluding region of the at least one object-excluding regionare formed by a same channel of the channel structure.
 12. The method ofclaim 11, wherein each object-excluding region of the at least oneobject-excluding region is continuously contiguous with theobject-accessible region between the upstream section and the downstreamsection.
 13. The method of claim 9, wherein the object-accessible regionincludes an object-accessible groove, and wherein the at least oneobject-excluding region includes one or more wings located adjacent theobject-accessible groove.
 14. The method of claim 9, wherein the atleast one object-excluding region includes a pair of object-excludingregions that are separated from one another by the object-accessibleregion.
 15. The method of claim 1, wherein the object-accessible regionis formed by a primary channel of the channel structure, wherein the atleast one object-excluding region includes one or more by-pass channelsdefined by the channel structure, and wherein the step of movingincludes a step of moving at least part of the portion of the carrierfluid to the one or more by-pass channels.
 16. The method of claim 1,wherein the objects are selected from the group consisting of beads,droplets, and biological cells.
 17. The method of claim 1, furthercomprising partitioning a stream including the carrier fluid andcarrying objects of the set at a position downstream of the downstreamsection to form isolated volumes, wherein partitioning includesencapsulating the isolated volumes with a liquid that is immiscible withthe carrier fluid.
 18. The method of claim 17, wherein the objectsinclude biological cells, wherein the stream includes beads, and whereineach isolated volume of a plurality of the isolated volumes containsonly one of the beads and only one of the biological cells.
 19. A systemfor arranging a set of objects, comprising: a microfluidic channelstructure having a reformatting zone including an object-accessibleregion and at least one object-excluding region; and at least one sourceof positive/negative pressure operatively connected to the channelstructure and configured to form a stream of carrier fluid transportingobjects of the set in the channel structure; wherein the channelstructure is configured such that a portion of the carrier fluid ismoved from the object-accessible region to the at least oneobject-excluding region in an upstream section of the reformatting zone,to reduce a spacing between objects of the set in the upstream section,and such that the portion of the carrier fluid is directed into theobject-accessible region from the at least one object-excluding regionin a downstream section of the reformatting zone, to increase a spacingbetween objects of the set in, and/or downstream of, the downstreamsection.